Cytochrome P-450 is known to play vital roles in the biotransformation of steroid hormones, fatty acids, drugs and carcinogens. This project is proposed to study the molecular properties, catalysis and mechanisms of carcinogen activation of this enzyme. Cytochrome P-450 with the highest possible purity will be prepared from rat liver microsomes. The molecular properties and the possible differences between P-450 and P-448 will be studied with respect to electrophoretic mobilities, N-terminal amino acids, peptide mapping, amino acid compositions, and numbers of sulfhydryl groups. The substrate and inhibitor binding sites will be studied with chemical labeling and enzyme kinetic techniques. Reversible cross-linking techniques will be used to study the molecular associations of the mono-oxygenase system. The interactions between purified cytochrome P-450 and microsomes will be studied to shed light on the molecular organizations and the mechanisms of electron transfer of the mono-oxygenase. The mode of inhibition of the mono-oxygenase activities by butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate will be studied with the reconstituted mono-oxygenase system. The mechanisms of carcinogen activation will be investigated by 1) determining the lifetime and properties of the "activated" metabolites of benzpyrene, 2) using the reconstituted mono-oxygenase system to examine the possible involvement of other microsomal components in carcinogen activation, and 3) studying the interactions between benzpyrene and nuclear components and testing our hypothesis that carcinogens are preferentially activated at the nuclear membrane.